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Metagenomic Analysis of Fever, Thrombocytopenia and Leukopenia Syndrome (FTLS)
What is FTLS? There have been numerous cases of fever, thrombocytopenia and leucopenia syndrome (FTLS) in Henan Province, China since 2007. Several of these cases involve tick bites. This suggests that infection might contribute to FTLS. Therefore, many microbial pathogens, which are transmitted by ticks, were tested via PCR/RT-PCR and indirect immunofluorescense assays (IFA). Only 8% of the collected samples tested positive for granulocytic anaplasmosis (HGA). This suggested that other pathogens were most likely involved, so an unbiased metagenomic screening and surveying method was used to study other microbes that may have been associated with FTLS. A new bunyavirus to identity to Tehran virus was discovered via BLASTx analysis of deduced protein sequences. It was present only in sera that were taken from FTLS patients. Furthermore, a phylogenetic analysis showed that this virus was distinct, even though it was closely related to Uukuniemi virus of Phleobvirus genus. The candidate virus was tested for association with FTLS along with samples that were colleted from Henan province during 2007 to 2010. Results showed that about 78% of acute-phase serum samples contained the viral DNA, and 73 of the 95 patients who had available acute and convalescent sera had evidence of infection. This new virus was isolated and named Henan Fever Virus (HNF). It is a new bunyavirus that almost identical to Uukuniemi virus. It is also strongly correlated with FTLS. Methods In May 2007, a county hospital in XXinyan City, Henan Province treated patients with fevers with the following characteristics: acute onset with fever, low white blod cell and platelet counts, high levels of alanine and aspartate transaminases, and positive urine protein. Based on epidemiological and clinical characteristics, two types of diseases were possible. These were rickettsial and arthropod-borne viral disease. Howeverm PCR assay results had no positive matches, which suggested a new infections agent. The research team adopted the following strategy to identify the infectious agent: 1) Sequencing randomly amplified cDNA/DNA from FTLS patient samples (using high throughput illumina sequencing to research viral communities that were present in the patients) 2) PCR detection of target DNA directly from clinical specimens 3) Viral culture 4) Immunodetection methods 5) Electron microscopic study of the morphology of the cultured virus Results All the patients with FTLS who were used for research were from the Henan Province of China. They were diagnosed based on the symptoms and clinical manifestations mentioned above. All the patients represent four different epidemiologically linked sporadic cases from 2007 to 2010. Most of the outbreaks occurred during the months between April and October. The peak was in April-May, which was the tea picking season in Henan. All of the patients lived in mountainous and hilly rural areas. 238 of the 285 tested patients were positive for the new bunyavirus infection, which was done via RT-PCR and IFA. The median age of affected patients was 57.2 years, and the range was 23-88. About 92% of the patients were farmers. The other 8% consisted of workers and students. 22% of the patients reported a tick bite within 2 weeks before the onset of symptoms. The rest of the patients did not remember getting a tick bite. Most patients had the following symptoms: sudden fever that lasted up to 10 days, fatigue, anorexia, headache, myalgia, arthalgia, dizziness, enlarged lymph nodes, muscle aches, vomiting and diarrhea. Some of the patients had more serious complications, such as hypotension, mental status alterations, and ecchymosis. However, most patients had positive outcomes. Further tests showed that patients had higher levels of lactate dehydrogenase, creatine kinase, AST and ALT enzymes.